香鱼抗菌肽LEAP-2的作用及机制研究

Liver-expressed antimicrobial peptide 2 (LEAP-2) is one of the cationic antimicrobial peptides and extremely important components of the innate immune system found in animals. Literatures reveal LEAP-2 has a direct antibacterial activity against a variety of microorganisms, and may relate to the activation of immune cells. However, its function and mechanism of action in fish remain unclear. Based on our preliminary research foundation, we aim to study the dual role of ayu (Plecoglossus altivelis) LEAP-2 in killing bacteria directly and mediating of immune cells. By examining the effect of LEAP-2 on the antibacterial activity, the DNA binding ability, the membrane damage and ultrastructure change of bacteria in vitro, we will illustrate the mechanism of direct bacterial killing. By observing the survival, bacterial load in tissue, pathological characteristic and cytokine expression in infected fish treated with LEAP-2, we will illustrate the protective immunity effect of LEAP-2. By using primary cultured head kidney-derived monocytes/macrophages, we will investigate the activation effect of LEAP-2 on monocytes/macrophages including the alterations of phagocytosis, bacterial killing, and gene expression profiles to illustrate whether LEAP-2 influence the activity of monocytes/macrophages. This work will not only reveal immune modulation mechanism of fish antimicrobial peptides, but also lay the theory foundation for research and development of antimicrobial peptides as new drug or agent.

肝表达抗菌肽2 (LEAP-2)是阳离子抗菌肽的一种，是动物先天免疫的重要组成部分。文献揭示，动物LEAP-2除具有直接杀菌活性外，还可能与免疫细胞激活有关，但鱼类中作用及机制均不清楚。基于前期研究基础，本项目拟探讨香鱼LEAP-2直接杀菌和调控免疫细胞的双重作用及相应机制。拟体外研究LEAP-2的抗菌活性以及对DNA结合能力、细菌细胞膜的损伤与超微结构的影响，初步阐明LEAP-2的直接抗菌作用机制；整体动物水平上研究LEAP-2对感病香鱼存活率、组织载菌量、组织病理和细胞因子表达的影响，明确LEAP-2对感病香鱼的免疫保护作用；体外细胞培养研究LEAP-2对香鱼单核/巨噬细胞吞噬与杀菌能力、基因表达谱的影响，初步阐明LEAP-2是否影响单核/巨噬细胞的活性。研究结果不仅能揭示鱼类抗菌肽参与机体免疫反应的调控机制，而且能为抗菌肽类新型药物或制剂的研发提供理论依据。

肝表达抗菌肽2 (LEAP-2)是阳离子抗菌肽的一种，是动物先天免疫的重要组成部分。本研究首先化学合成香鱼LEAP-2成熟肽用于测定其直接杀菌作用，结果表明，香鱼LEAP-2对鳗利斯顿氏菌和迟缓爱德华氏菌的抗菌活性最强，其次是大肠杆菌；扫描电镜和透射电镜观察发现，经LEAP-2处理的细菌表面出现皱缩，有明显孔洞，原生质出现渗漏；琼脂糖凝胶电泳检测发现，LEPA-2与细菌基因组DNA或质粒DNA孵育后出现水解；渗透率测定结果表明，LEPA-2能引起细菌细胞膜的通透性显著增强。揭示香鱼LEAP-2可能通过与细菌细胞膜结合，使细胞膜通透性增强，破坏细胞膜结构，在细胞膜上形成许多孔道，造成细菌胞内原生质渗漏，影响细菌代谢系统，进而膜局部破损，最终引起细胞膜破碎，细胞完全解体，从而起到抑菌与杀菌作用。其次，鉴于香鱼LEAP-2基因mRNA的表达与鳗利斯顿氏菌感染密切相关，研究了LEAP-2对香鱼的免疫保护作用，结果发现，LEAP-2蛋白通过增强鳗利斯顿氏菌感染香鱼的存活率、降低组织载菌量及促炎细胞因子表达来发挥免疫作用。进一步从细胞水平上研究发现，LEAP-2能够增强单核/巨噬细胞吞噬与杀菌能力以及呼吸爆发功能，抑制细菌感染诱导的促炎细胞因子表达。最后以LEAP-2为诱饵蛋白，从香鱼单核/巨噬细胞cDNA文库中筛选互作蛋白，最终获得一个名为癌胚抗原相关的细胞粘附分子20 (Carcinoembryonic antigen-related cell adhesion molecule 20, CEACAM20) 的蛋白。综上，香鱼LEAP-2不仅具有直接抑菌与杀菌作用，并可通过激活香鱼单核/巨噬细胞的功能发挥机体免疫保护作用。本研究在国际上首次系统阐明抗菌肽LEAP-2参与鱼类细菌性感染免疫应答中的作用及机制，为抗菌肽类新型药物或制剂的研发奠定了理论基础。